[Expression of human spindle mitosis arrest deficiency gene in spontaneous abortion embryo tissues].
OBJECTIVE
To investigate the expression of human spindle mitosis arrest deficiency gene (hsMAD2) in spontaneous abortion embryos and the relationship between low expression of hsMAD2 and numerical chromosomal aberration. METHODS Spontaneous abortion embryo tissues were collected, including 23 cases of once spontaneous abortion tissue and 10 cases of twice or more spontaneous abortion tissue and induced abortion embryos (35 cases) from the Department of Gynaecology and Obstetrics of the Affiliated Hospitals of Chongqing University of Medical Science during the period of March 2006 to March 2007. FQ-PCR and western blot were used to evaluate the endogenous expression level of hsMAD2 mRNA and hsMAD2 protein; primary culturing of cells from the induced abortion embryos was conducted and 5 embryonic cells were selected by chromosomes karyotype analysis. Recombinant shRNA plasmids targeting hsMAD2 gene were constructed to inhibit the expression of endogenous hsMAD2 genes in embryonic cells which have normal karyotypes; the groups were defined as the first experimental group (transfected with pshRNA-hsMAD2-1) , the second experimental group (transfected with pshRNA-hsMAD2-2), the third experimental group (transfected with pshRNA-hsMAD2-3), the first control group (transfected with nothing), the second control group (transfected with pTZU6 + 1) and the independent group (transfected with pshRNA-N1). Interference efficiency was demonstrated by FQ-PCR and western blot; cell proliferation was measured by methyl thiazolyl tetrazolium (MTT) assay; cell-cycle was assessed by flow cytometry (FCM); the chromosome numbers were calculated to analyze the variation of chromosomes.


RESULTS
(1) The mRNA levels of hsMAD2 in the once spontaneous abortion tissue, twice or more spontaneous abortion tissue and induced abortion tissue were 0.00879 +/- 0.00035, 0.00901 +/- 0.00033 and 0.00941 +/- 0.00026 respectively, and there was no significant difference (P > 0.05) compared with each other; however, the protein levels of hsMAD2 in three groups were 0.2791 +/- 0.0311, 0.0431 +/- 0.0020 and 0.5790 +/- 0.0331 respectively, and there were significant differences (P < 0.05) compared with each other. (2) Recombinant shRNA plasmids could significantly and specifically inhibit hsMAD2 gene expression in embryonic cells. Compared with the first control group (4%) and the second control group (3%), the recombinant shRNA could inhibit embryonic cell proliferation to 54% at 48 h after transfection (P < 0.05); compared with the first control group (8.2%) and the second control group (8.0%), the ratios of G2/M phase cells in the experimental group (17.9%)was significantly increased (P < 0.05); compared with the first control group (4.8%), the ratios of abnormal chromosomes in the experimental group was increased to 30.0% (P < 0.05).


CONCLUSIONS
Down-expression of hsMAD2 gene may be one of the mechanisms inducing numerical chromosome aberration, abnormal embryo development and the occurrence of spontaneous abortion.
Cai
Yan
Y
Key Laboratory of Ministry of Education, the Faculty of Laboratory Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China.
Wang
Jian
J
Yuan
Tai-Xian
TX
Shi
Qiong
Q
Weng
Ya-Guang
YG
Wang
Ying-Xiong
YX
Jiang
Hong-Yan
HY
Liu
Zi-Jie
ZJ
D000028:Abortion, Induced; D000022:Abortion, Spontaneous / Q000378:metabolism* / Q000473:pathology; D000328:Adult; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D002869:Chromosome Aberrations; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D064246:Mad2 Proteins; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins